Lantibiotics form a group of unique ribosomally synthesized and posttranslationally modified antibiotic peptides that are produced by, and primarily act on, Gram-positive bacteria (for review, See McAuliffe et al., FEMS Microbiol. Rev. 25: 285-308 (2001)). Because by definition lantibiotics contain intramolecular thioether bridges or rings formed by the thioether amino acids lanthionine (Lan) and 3-methyllanthionine (MeLan) and lantibiotics all are peptide antibiotics with moderate to strong bactericidal activity, lantibiotics take their name from these most eye-catching properties.
Thioether rings protect peptides against proteolytic degradation. For instance, the lantibiotic nisin remains active after trypsin treatment. Thioether rings are also essential for some lantibiotic activities. For instance, opening of ring A or C in nisin causes deletion of the membrane permeabilization capacity. Ring A of nisin is necessary for its capacity to autoinduce its own synthesis and for nisin's capacity to block the peptidoglycan synthesis by interacting with lipid II. It is essential to have thioether rings and not disulfide rings since replacement of thioether rings by disulfide bridges leads to loss of antimicrobial activity.
Lantibiotics do not spoil the environment and are not toxic to animals or man. Lantibiotics may also have applications as biopreservatives in the preparation of food and beverages, or as a bactericidal agent in cosmetics and veterinary and medical products. Because the growing number of multidrug resistant pathogenic micro-organisms has created the threat of another “pre-antibiotic era” for many bacterial diseases, it is expected that lantibiotics also may serve as new lead compounds to remedy this alarming problem. For these reasons, lantibiotics have experienced a marked increase in basic and applied research activities in the past decade, leading to an extraordinary increase in the knowledge of the lantibiotics structural and functional properties, the mechanisms of action and of the genes and protein components involved in the biosynthesis and secretion of the lantibiotics. For example, lantibiotics have become subject to “protein engineering” projects, with the aim of altering, via site-directed mutagenesis, the lantibiotics activity, stability and spectrum of susceptible target cells. In this description, the linear (type A) lantibiotics are considered since at present, very little specific information is available for the circular (type B) lantibiotics.
The lantibiotics subtilin and nisin belong to, and are representative of, the peptide antibiotics or lantibiotics of type A. Subtilin and nisin contain the rare amino acids dehydroalanine (Dha), dehydrobutyrine (Dhb), meso-lanthionine and 3-methyllanthionine, as well as the characterizing thioether bridges.
Nisin is the most prominent lantibiotic and is used as a food preservative due to its high potency against certain Gram-positive bacteria. It is produced by Lactococcus lactis strains belonging to serological group N. The potent bactericidal activities of nisin and many other lantibiotics are based on their capacity to permeabilize the cytoplasmic membrane of target bacteria. Breakdown of the membrane potential is initiated by the formation of pores through which molecules of low molecular weight are released. In addition, nisin inhibits cell wall synthesis by binding to lipid II, a precursor of peptidoglycan synthesis, modulates the activity of autolytic enzymes and inhibits the outgrowth of spores (See also, Breukink and de Kruijff, Biochem. Biophys. Acta 1462: 223-234, 1999).
In several countries, nisin is used to prevent the growth of clostridia in cheese and canned food. The nisin peptide structure was first described by Gross & Morell (J. Am. Chem. Soc 93: 4634-4635, 1971), and its structural gene was isolated in 1988 (Buchmann et al., J. Biol. Chem. 263: 16260-16266, 1988). Nisin has two natural variants, nisin A and nisin Z, which differ in a single amino acid residue at position 27 (histidine in nisin A is replaced by asparagine in nisin Z).
Subtilin is produced by Bacillus subtilis (ATCC 6633). Subtilin's chemical structure was first unravelled by Gross & Kiltz (Biochem. Biophys. Res. Commun. 50: 559-565, 1973) and its structural gene was isolated in 1988 (Banerjee & Hansen, J. Biol. Chem. 263: 9508-9514, 1988). Subtilin has strong similarities to nisin with an identical organization of the lanthionine ring structures (FIG. 1). Further, nisin and subtilin possess similar antibiotic activities.
Due to its easy genetic analysis B. subtilis became a suitable model organism for the identification and characterization of genes and proteins involved in lantibiotic biosynthesis. The pathway by which nisin is produced is very similar to that of subtilin and the proteins involved share significant homologies over the entire proteins (for review, see also, De Vos et al., Mol. Microbiol. 17: 427-437, 1995).
Another well known and studied lantibiotic, produced by Staphylococcus epidermis 5, is Pep 5, which contains three ring structures (one MeLan and two Lan), an N-terminal oxobutyryl residue and two Dhb residues (Kellner et al., Angew. Chemie Int. Ed. Engl. 28: 616-619, 1989).
The respective genes of subtilin and nisin have been identified adjacent to the structural genes, and are organized in operon-like structures (FIG. 2). The genes are thought to be responsible for posttranslation modification, transport of the modified prepeptide, proteolytic cleavage and immunity which prevents toxic effects on the producing bacterium. Further, biosynthesis of subtilin and nisin is strongly regulated by a two-component regulatory system which includes a histidine kinase and a response regulator protein.
According to a present model (FIG. 3), it is assumed that an extracellular growth phase-dependent signal may activate the membrane localized histidine kinase. The nature of the signal may be different for subtilin and nisin biosynthesis. In nisin biosynthesis, nisin has an inducing function, whereas it was shown for subtilin biosynthesis that its biosynthesis is sporulation dependent.
According to the model, after nisin's auto-phosphorylation, the SpaK and NisK histidine kinase transfer the phosphate residue to the response regulator which in turn activates the genes necessary for subtilin and nisin biosynthesis. Thereafter, the prepeptide is modified at a membrane localized modification complex (lantionine synthetase) including the intracellular SpaB/SpaC and the NisB/NisC proteins, respectively. According to the model, the proteins are also associated with the SpaT and the NisT transporter, respectively.
As in any lantibiotic, the presubtilin or prenisin molecule includes a leader segment and a mature segment, wherein the leader segment is thought to play several roles in the biosynthetic pathway. The leader segment is thought not to be just a translocation signal sequence, but also thought to provide recognition signals for the modification enzymes and to suppress antimicrobial activity until the mature peptide is released from the cell. As also postulated by Qiao and Saris, (Fems Microbiol. Let. 144: 89-93(1996)), the modified prepeptide is thought to be proteolytically cleaved after its transport through the cellular membrane or cleavage of the leader from the modified peptide occurs inside the cell before secretion. In the case of nisin, cleavage is performed by NisP, whereas in the case of subtilin, no specific protease has been found within the operon-like structure. However, B. subtilis is rich in extracellular proteases and possibly subtilisin, which also recognizes proline at position-2, could cleave the modified pre-subtilin.
The gene clusters flanking the structural genes for various linear (type A) lantibiotics have recently been characterized (for review, see, Siezen et al., Antonie van Leeuwenhoek 69: 171-184, 1996)). The best studied representatives are those of nisin (nis), subtilin (spa), epidermin (epi), Pep5 (pep), cytolysin (cyl), lactocin S (las) and lacticin 481 (lct). Comparison of the antibiotic gene clusters shows that the clusters contain conserved genes that probably encode similar functions.
The nis, spa, epi and pep clusters contain lanB and lanC genes that are presumed to code for two types of enzymes that have been implicated in the modification reactions characteristic of all antibiotics, i.e. dehydration and thio-ether ring formation. The cyl, las and lct gene clusters have no homologue of the lanB gene, but do contain a much larger lanM gene that is the lanC gene homologue. Most antibiotic gene clusters contain a lanP gene encoding a serine protease that is presumably involved in the proteolytic processing of the prelantibiotics. All clusters contain a lanT gene encoding an ATP-binding cassette (ABC)-like transporter spanning the plasma membrane of a cell and likely is involved in the export of (precursors of) the lantibiotics from the cell. The lanE, lanF and lanG genes in the nis, spa and epi clusters encode another transport system that is possibly involved in self-protection. In the nisin and subtilin gene clusters two tandem genes, lanR and lank, have been located that code for a two-component regulatory system.
Non-homologous genes are also found in some lantibiotic gene clusters. The nisL and spaI genes encode lipoproteins that are involved in immunity, the pepL gene encodes a membrane-located immunity protein and epiD encodes an enzyme involved in a posttranslational modification found only in the C-terminus of epidermin. Several genes of unknown function are also found in the las gene cluster. Commonly, a host organism or cell carrying one or more of the genes (e.g., lanT, lanI, lanA, lanP, lanB and lanC) in the cluster are identified with a shorthand notation such as lanTIAPBC. The above identified genes appear to be different from genes encoding the secretion apparatus for the non-lantibiotic lactococcins that includes two membrane proteins LcnC and LcnD, as discussed in Franke et al., J. Biol. Chem 274: 8484-8490, (1999).
A database has been assembled for all putative gene products of type A lantibiotic gene clusters. Database searches, multiple sequence alignment and secondary structure prediction have been used to identify conserved sequence segments in the LanB, LanC, LanE, LanF, LanG, LanK, LanP, LanM, LanR and LanT gene products that may be essential for structure and function (Siezen et al., ibid). The database allows for a rapid screening of newly determined sequences in lantibiotic gene clusters.
However, despite the above cited recent knowledge obtained in the field, attempts to engineer novel lantibiotic-like peptides comprising newly synthesized unnaturally occurring thioether bridges have been scarce, if not rather unsuccessful. In U.S. Pat. No. 5,861,275, nisin-subtilin chimeras have been produced in the Gram-positive Bacillus subtilus that do not comprise thioether bridges other than those naturally occurring in either nisin or subtillin. In a different application (U.S. Patent Application Publication 2002/0019518), Bacillus subtilus was used to produce a chimeric polypeptide comprising a lantibiotic peptide and a subtilin leader segment, a lantibody, that remains associated within the cell wall.
A novel thioether bridge in lantibiotic Pep5 has been engineered by Bierbaum et al., Appl. Env. Microbiol. 62: 385-392, (1996) by modifying the Gram-positive bacterium Staphylococcus epidermis 5 by depleting the host organism of the gene cluster pepTIAPBC and replacing pepTIAPBC with a gene cluster pepIAPBC, wherein pepA was or was not replaced with mutated structural genes encoding for Pep 5 peptide and wherein amino acids were substituted; genes coding for peptides with substitutions C27A (Cysteine to Alanine at position 27), C33A, A19C, Dhb16A, Dhb20A, K18Dha were generated. The A19C substitution resulted in novel thioether ring formation. The clone corresponding to the A19C substitution produced a small amount of a peptide that showed only little activity. It was thought that prolonged exposure of the peptide to intracellular protease of the producing transformed cell was causal to this disappointing result.
The K18Dha substitution in Pep5 resulted in a clone that produced incompletely dehydrated serine at position 18. Kuipers et al. (J. Biol. Chem. 267: 24340-24346, 1992) engineered a new Dhb residue into nisinZ by substituting M17Q/G18T in the lantibiotic, but also obtained incomplete dehydration of the resulting threonine and no additional ring formation. The incomplete dehydration is generally thought to be a result of questionable substrate specificity of the dehydrating enzyme LanB in the transformed cell.
In short, no large measure of success has yet been achieved in providing novel thioether bridges to lantibiotics in Gram-positive organisms, let alone that engineered thioether bridge formation has been provided to polypeptides of non-lantibiotic descent or by organisms other than Gram-positive bacteria.
Paul, Leena K et al. (FEMS Microbiol. Lett. 176: 45-50, 1999) recently studied the subtilin leader peptide as a translocation signal in the Gram-negative E. coli. By default, devoid of a specific lantibiotic transporter system and provided a fusion-protein comprising the subtilin leader peptide and part of the mature subtilin attached to E. coli alkaline phosphatase (AP) to study the possible translocation. Although the fusion protein was translocated to the periplasmic side of the cytoplasmic membrane, the fusion protein remained associated with that membrane. In earlier work (Izaguirre & Hansen, Appl. Environ. Microbiol 63: 3965-3971, 1997), the same fusion protein was expressed in the Gram-positive Bacillus subtilis, where the fusion protein was cleaved off from the membrane after successful translocation, but where no dehydration of serines or threonines of the AP polypeptide, let alone thioether bridge formation, was observed. Novak J et al., ASM general meeting 96: 217 (1999), recently provided an E. coli host cell with an ORF (ORF1) encoding an ABC transporter of 341 amino acids thought to be involved in the translocation of the lantibiotic mutacinII in Streptococcus mutans. However, an intact gene product of the ORF1 was not produced in E. coli, whereas a truncated protein of unknown identity or functionality was observed.
For the purpose of protein engineering of lantibiotics (for an extensive review see, Kuipers et al., Antonie van Leeuwenhoek 69: 161-170, 1996) or for the purpose of engineering newly designed (poly)peptides with lantibiotic-type posttranslational modifications, i.e., for example, for pharmaceutical use, much attention has recently (see, e.g., Entian & de Vos, Antoni van Leeuwenhoek supra; Siegers et al., J. Biol. Chem. 271: 1294-12301 (1996); Kiesau et al., J. Bacter. 179: 1475-1481 (1997)) been given to understanding the role of the LanB, LanC (or LanM) and LanT complex, the enzymes thought to be involved in dehydration, thioether ring formation and transportation of the lantibiotic out of the cell, respectively. However, the production of lantibiotic type heterologous proteins has not yet matured satisfactorily.